This invention relates to substituted benzo[1,2-b:5,4-bxe2x80x2]dipyran4-amines which are modulators, agonists or antagonists, of the CC chemokine receptor CC-CKR5 now designated as CCR5 (Nature Medicine, 2: 1174-8, 1996). In addition, this invention relates to the treatment and prevention of disease states mediated by CCR5.
T cells are not only key regulators of the immune response to infectious agents but are believed critical for the initiation and maintenance of the inflammatory reaction in a variety of chronic diseases. Increased numbers or enhanced activation state of T cells, especially CD4+ T cells, have been demonstrated in the synovium of individuals with rheumatoid arthritis (M. J. Elliott and R. N. Maini, Int. Arch. Allergy Immunol. 104: 112-1125, 1994), in the bronchial mucosa of asthmatics (C. J. Corrigan and A. B. Kay, Immunol. Today 13: 501-506, 1992), in the lesions of multiple sclerosis (R. Martin and H. F. McFarland, Crit. Rev. Clin. Lab. Sci. 32: 121-182, 1995), in psoriatic lesions (J. L. Jones, J. Berth-Jone, A. Fletcher and P. E. Hutchinson, J. Pathol. 174: 77-82, 1994) and in the fatty streaks of atherosclerosis (R. Ross, Annu. Rev. Physiol. 57: 791-804, 1995).
T cells, as well as other inflammatory cells, will migrate into tissues in response to the production of a variety chemotactic factors. Among these factors are a superfamily of 8-12 kDa proteins known as the chemokines. These proteins share structural features such as the presence of 3-4 conserved cysteine residues. RANTES, which stands for Regulated upon Activation Normal T cell Expressed and Secreted, is a 8 kDa protein member of CC branch of the chemokine family. These proteins recruit and activate immune and inflammatory cells through an interaction with G-protein coupled receptors. The CC branch is defined by the absence of an intervening amino acid residue between the first two cysteine residues and members of this family predominately elicit the migration of mononuclear cells, eosinophils and basophils (M. Baggiolini, B. Dewald, and B. Moser, Adv. Immunol. 55: 97-179, 1994; and J. J. Oppenheim, C. O. C. Zachariae, N. Mukaida, and K. Matsushima, Annu. Rev. Immunol. 9: 617-648, 1991).
RANTES potently produces chemotaxis of T cells, basophils, eosinophils, monocytes and mast cells. RANTES was originally identified as gene product induced late after antigen activation of T-cells (T. J. Schall, J. Jongstra, B. J. Dyer, J. Jorgensen, et al., J. Immunol. 141:1018-1025, 1988), however, RANTES has been shown to be synthesized and secreted by a diverse group of cells that include epithelial and endothelial cells (C. Stellato, L. A. Beck, G. A. Gorgone, D. Proud, et al., J. Immunol. 155: 410-418, 1995; and A. Marfaing-Koka, O. Devergne, G. Gorgone, A. Portier, et al., J. Immunol. 154: 1870-1878, 1994), synovial fibroblasts (P. Rathanaswami, M. Hachicha, M. Sadick, T. J. Schall, et al., J. Biol. Chem. 268: 5834-5839, 1993) and dermal fibroblasts (M. Sticherling, M. Kupper, F. Koltrowitz, E. Bornscheuer, et al., J. Invest. Dermatol. 105: 585-591, 1995), mesangial cells (G. Wolf, S. Aberle, F. Thaiss, et al., Kidney Int. 44: 795-804, 1994) and platelets (Y. Koameyoshi, A. Dorschner, A. I. Mallet, E. Christophers, et al., J. Exp. Med. 176: 587-592, 1992). In these cells RANTES mRNA is rapidly upregulated in response to IL-1 or TNFxcex1. Although RANTES mRNA is not usually detected in normal tissues (J. M. Pattison, P. J. Nelson, and A. M. Krensky, Clin. Immunother. 4: 1-8, 1995), increased mRNA or protein has been found in diseases characterized by a mononuclear infiltrate. For example, RANTES mRNA was visualized using in situ hybridization in renal allografts undergoing rejection (J. M. Pattison, P. J. Nelson, and A. M. Krensky, Clin. Immunother. 4: 1-8, 1995; and K. C. Nadeau, H. Azuma and N. I. Tilney, Proc. Natl. Acad. USA 92: 8729-8733, 1995) in the skin of a topic dermatitis patients after exposure to antigen (S. Ying, L. Taborda-Barata, Q. Meng, M. Humbert, et al., J. Exp. Med. 181: 2153-2159, 1995), and in endothelial cells of coronary arteries undergoing accelerated atherosclerosis after cardiac transplant (J. M. Pattison, P. J. Nelson, and A. M. Krensky, Clin. Immunother. 4: 1-8, 1995). Further, increased immunoreactive protein for RANTES has been detected in bronchoalveolar lavage fluid (R. Alam, J. York, M. Boyers, et al., Am. J. Resp. Crit. Care Med. 149: A951, 1994) and sputum from asthmatic individuals (C. M. Gelder, P. S. Thomas, D. H. Yates, I. M. Adcock, et al., Thorax 50: 1033-1037, 1995).
Several receptors have been identified that bind RANTES. In particular, CCR5, when expressed in either HEK 293 cells or CHO cells, binds RANTES. This receptor is expressed in T-cells and in monocytes and macrophages, immune/inflammatory cells which are important in the maintenance of a chronic inflammatory reaction. Pharmacological characterization of CCR5 indicates sirnilarities to the RANTES binding site observed on isolated T cells. Therefore, antagonism of RANTES"" action on CCR5, as well as antagonism of other natural modulators of CCR5, should inhibit the recruitment of T cells into inflammatory lesions and provide a novel therapeutic approach for the treatment of a topic and autoimmune disorders.
Since T cells express CCR5, selective receptor modulators of CCR5, particularly antagonists, are likely to provide beneficial effects in diseases including, but not limited to, asthma and a topic disorders (for example, a topic dermatitis and allergies), rheumatoid arthritis, atherosclerosis, psoriasis, sarcoidosis and other fibrotic diseases, autoimmune diseases such as multiple sclerosis, and inflammatory bowel disease, all in mammals, preferably humans. Furtermore since CD8+ T cells have been implicated in COPD, CCR5 may play a role in their recruitment and therefore antagonists to CCR5 could provide potential therpeutic in the treatment of COPD. Also since CCR5 is a co-receptor for the entry of HIV into cells, selective receptor modulators may be useful in the treatment of HIV infection.
Surprisingly, it has now been discovered that a class of non-peptide compounds, in particular substituted benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amines of formula (I), function as CCR5 receptor modulators, and therefore, have utility in the treatment and prevention of disease states mediated by CCR5 receptor mechanisms.
In one aspect, the present invention is to a genus of novel compounds of formula (I), or pharmaceutically active salts thereof, said compounds which are also useful in treating the above-mentioned CCR5-mediated disease states: 
wherein:
R1, R2, R6, and R7 are independently hydrogen or C1-6alkyl;
R3 is hydrogen or hydroxy;
R4 is hydrogen or C1-6alkyl;
R5 is hydrogen, C1-6alkyl, C3-6cycloalkyl, or aralkyl; or, NR4R5 may form a heterocyclic ring having 5-, 6-, or 7-members, optionally containing one of oxygen, sulfur, or NR8;
R8 is hydrogen, C1-6alkyl, or aralkyl;
X is hydrogen or one or more of C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, C5-6cycloalkenyl, aralkyl, aryl, CH2NR9R10, CH2OR11, COR11, CONR9R10, CO2R11, cyano, trifluoromethyl, NR9R10, NR9COR11, NR9CONR9R10 NR9CO2R12, NR9SO2R13, nitro, hydroxy, C1-6alkoxy, OC(O)R11, OC(O)NR9R10, SR14, SOR13, SO2R13, SO2NR9R10 or halogen;
R9 and R10 are independently hydrogen, C1-6allkyl, aralkyl or aryl; or NR9R10 forms a heterocyclic ring having 5-, 6-, or 7-members, optionally containing one oxygen, sulfur, or NR8;
R11 is hydrogen, C1-6alkyl, aralkyl, aryl, or trifluoromethyl;
R12 is C1-6alkyl, aralkyl, aryl, or trifluoromethyl;
R13 is C1-6alkyl, aralkyl, aryl, or trifluoromethyl;
R14 is hydrogen, C1-6alkyl, aralkyl, aryl, or trifluoromethyl; and  is a single or a double bond.
In another aspect, the present invention is to a method of treating CCR5 mediated disease states, including, but not limited to, COPD, asthma and a topic disorders (for example, a topic dermatitis and allergies), rheumatoid arthritis, sarcoidosis and other fibrotic diseases, atherosclerosis, psoriasis, autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, and HIV infection, all in mammals, preferably humans, comprising administering to such mammal in need thereof, a benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine of formula (I), or pharmaceutically active salts thereof.
In yet another aspect, the present invention is to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier therefor. In particular, the pharmaceutical compositions of the present invention are used for treating CCR5-mediated disease states, including, but not limited to,COPD asthma and a topic disorders (for example, a topic dermatitis and allergies), rheumatoid arthritis, sarcoidosis and other fibrotic diseases, atherosclerosis, psoriasis, autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, and HIV infection.
It has now been discovered that substituted benzo[1,2-b:5,4bxe2x80x2]dipyran-4-amines of formula (I) are CCR5 receptor modulators. It has also now been discovered that selective inhibition of CCR5 receptor mechanisms by treatment with the receptor modulators of formula (I), or a pharmaceutically acceptable salt thereof, represents a novel therapeutic and preventative approach to the treatment of a variety of disease states, including, but not limited to, asthma and a topic disorders (for example, a topic dermatitis and allergies), rheumatoid arthritis, sarcoidosis and other fibrotic diseases, atherosclerosis, psoriasis, autoimmune diseases such as multiple sclerosis, and inflammatory bowel disease, all in mammals, preferably humans (xe2x80x9cCCR5-mediated diseasesxe2x80x9d). Furthermore, since CD8+ cells have been implicated in COPD. CCR5 may play a role in their recruitment and therefore antagonists to CCR5 could provide potential therpeutic in their recruitment and COPD. Also, since CCR5 is a co-receptor for the entry of HIV into cells, selective receptor modulators may be useful in the treatment of HIV infection.
The term xe2x80x9calkylxe2x80x9d is used herein at all occurrences to mean a straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like.
The terms xe2x80x9ccycloalkylxe2x80x9d and xe2x80x9ccyclic alkylxe2x80x9d are used herein at all occurrences to mean cyclic radicals, preferably comprising 3 to 6 carbon atoms which may be mono- or bicyclo-fused ring systems which may additionally include unsaturation, including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like.
The term xe2x80x9ccycloalkenylxe2x80x9d is used herein to mean cyclic radicals, preferably of 5 to 6 carbons, which have at least one double bond between two of the carbon atoms in the ring, including but not limited to cyclopentenyl, cyclohexenyl, and the like.
The term xe2x80x9calkenylxe2x80x9d is used herein at all occurrences to mean a straight or branched chain radical of 2 to 6 carbon atoms, unless the length is limited thereto, wherein there is at least one double bond between two of the carbon atoms in the chain, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like.
The term xe2x80x9calkynylxe2x80x9d is used herein at all occurrences to mean a straight or branched chain radical of 2 to 6 carbon atoms, unless the chain length is limited thereto, wherein there is at least one triple bond between two of the carbon atoms in the chain, including, but not limited to, acetylene, 1-propylene, 2-propylene, and the like.
The term xe2x80x9carylxe2x80x9d is used herein at all occurrences to mean 5-14-membered substituted or unsubstituted aromatic ring(s) or ring systems which may include bi- or tri-cyclic systems, including, but not limited to phenyl, naphthyl, and the like.
The term xe2x80x9caralkylxe2x80x9d is used herein at all occurrences to mean an aryl moiety as defined above, which is connected to a C1 to C6 alkyl moiety as defined above, for example, benzyl or phemethyl, and the like.
The term xe2x80x9calkoxyxe2x80x9d is used herein at all occurrences to mean a straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like.
The terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d are used interchangeably herein at all occurrences to mean radicals derived from the elements chlorine, fluorine, iodine and bromine.
The term xe2x80x9cheterocyclicxe2x80x9d is used herein at all occurrences to mean a saturated or partially unsaturated 5-, 6-, or 7-membered ring system (unless the cyclic ring system is otherwise limited) in which the ring optionally contains one other heteroatom selected from oxygen, sulfur, or NR8, wherein R8 is defined above, such as, but not limited to, piperazine or morpholine.
The term xe2x80x9cCCR5 mediated disease statexe2x80x9d is used herein at all occurrences to mean any disease state which is mediated (or modulated) by CCR5.
Suitably, pharmaceutically acceptable salts of formula (I) include, but are not limited to, salts with inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate, or salts with an organic acid such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, palmitate, salicylate, and stearate.
The compounds of the invention can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms, with pharmaceutically acceptable solvents such as water, ethanol, and the like, are equivalent to the unsolvated forms for purposes of this invention.
The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. The stereocenters may be of any combination of R and S configuration, for example, (R,R), (R,S), (S,S) or (S,R). All of these compounds are within the scope of the present invention.
For the compounds of formula (I) various embodiments are as follows.
X is suitably hydrogen or one or more of C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, C5-6cycloalkenyl, aralkyl, aryl, CH2NR9R10, CH2OR11, COR11, CONR9R10, CO2R11, cyano, trifluoromethyl, NR9R10, NR9COR11, NR9CONR9R10, NR9CO2R12, NR9SO2R13, nitro, hydroxy, C1-6alkoxy, OC(O)R11, OC(O)NR9R10, SR14, SOR13, SO2R13, SO2 NR9R10 or halogen. Preferably, X is hydrogen, trifluoromethyl, chloro, or methyl. More preferably, X is trifluoromethyl. When X is trifluoromethyl, X is preferably 3-CF3.
It will be understood that the substituent(s) X may be at any open position on the aromatic ring of formula (I) to which the substituent is attached. In addition, it will be understood that there may be more than one substituent X in any given compound of formula (I), and that if there is more than one substituent X, that substituent may be the same or different.
R1, R2, R6, and R7 are suitably, independently, hydrogen or C1-6alkyl. Preferably, R1, R2, R6, and R7 are C1-6alkyl. More preferably, R1, R2, R6, and R7 are meth
R3 is suitably hydrogen or hydroxy. Preferably R3 is hydrogen.
R4 is suitably hydrogen or C1-6alkyl and R5 is suitably hydrogen, C1-6alkyl, C3-6cycloalkyl, or aralkyl. Preferably, R4 is hydrogen and R5 is C1-6alkyl, C3-6cycloalkyl, or aralkyl, or R4 and R5 are C1-6alkyl. More preferably, R4 is hydrogen and R5 is methyl, cyclopropyl or cyclohexyl, or R4 and R5 are both methyl.
NR4R5 may suitably formn a heterocyclic ring having 5-, 6-, or 7-members, optionally containing one of oxygen, sulfur or NR8. When NR4R5 forms a ring, the preferred rings include morpholino and piperazinyl.
A preferred group of compounds of the invention is wherein.
X is hydrogen, trifluoromethyl, chloro or methyl;
R1 and R2 are methyl;
R6 and R7 are methyl;
R3 is hydrogen or hydroxy; and
R4 is hydrogen or C1-6alkyl, preferably methyl, and R5 is hydrogen, C1-6alkyl, C3-6cycloalkyl, or aralkyl; or NR4R5 forms a 5-7 membered ring, optionally containing one of oxygen, sulfur, or NR8.
A more preferred group of compounds of the invention is wherein:
X is trifluoromethyl;
R1 and R2 are methyl;
R6 and R7 are methyl;
R3 is hydrogen or hydroxy; and
R4 is hydrogen or methyl and R5 is hydrogen, methyl, cyclopropyl or cyclohexyl, or R4 and R5 are methyl, or NR4R5 forms a ring which is morpholino or piperazinyl.
Among the preferred compounds of the invention are the following compounds:
3,4,7,8-Tetrahydro-2,2,8,8-tetramethyl-4-morpholino-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran;
N-Cyclopropyl-3,4,7,8-tetrahydro-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran4-amine;
N-Cyclohexyl-3,4,7,8-tetrahydro-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine;
3,4,7,8-Tetrahydro-4-(4-methylpiperazin-1-yl)-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran;
N-Benzyl-3,4,7,8-tetrahydro-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine;
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzol[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride;
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine (isomer 1);
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2dipyran-4-amine (isomer 2)(;
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine (isomer 3);
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine (isomer 4);
3,4,7,8-Tetrahydro-N-methyl-2,2,8,8-tetramethyl-6-phenyl-2H,6H-benzo-[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride;
6-(3-Chlorophenyl)-3,4,7,8-tetrahydro-N-methyl-2,2,8,8-tetramethyl-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine;
6-(4-Chlorophenyl)-3,4,7,8-tetrahydro-N-methyl-2,2,8,8-tetramethyl-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride;
3,4,7,8-Tetrahydro-N-methyl-6-(4-methylphenyl)-2,2,8,8-tetramethyl-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine;
3,4,7,8-Tetrahydro-6-(4-methoxyphenyl)-N-methyl-2,2,8,8-tetramethyl-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine;
3,4,7,8-Tetrahydro-N,N-dimethyl-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride,
(4xcex1)-3,4,7,8-Tetrahydro-3xcex2-hydroxy-N,N-dimethyl-2,2,8,8-tetramethyl-6xcex2-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride hydrate; and
(4xcex2)-3,4,7,8-tetrahydro-3xcex1-hydroxy-N,N-dimethyl-2,2,8,8-tetramethyl-6xcex2-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran-4-amine hydrochloride.
Compounds excluded from the scope of this invention are as follows:
4-(4-Benzylpiperazin-1-yl)-3,4,7,8-tetrahydro-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo[1,2-b:5,4-bxe2x80x2]dipyran; and
N-tert-butyl-3,4,7,8-tetrahydro-2,2,8,8-tetramethyl-6-(3-trifluoromethylphenyl)-2H,6H-benzo-[1,2-b:5,4-bxe2x80x2]dipyran-4-amine.
The pharmaceutically effective compounds of this invention (and the pharmaceutically acceptable salts thereof) are administered in conventional dosage forms prepared by combining a compound of formula (I) (xe2x80x9cactive ingredientxe2x80x9d) in an amount sufficient to treat asthma and a topic disorders (for example, a topic dermatitis and allergies), rheumatoid arthritis, atherosclerosis, psoriasis, autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, and HIV infection, with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ngredients as appropriate to the desired preparation.
The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.
A wide variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1000 mg. When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
The active ingredient may also be administered topically to a mammal in need of treatment or prophylaxis of CCR5 mediated disease states. The amount of active ingredient required for therapeutic effect on topical administration will, of course, vary with the compound chosen, the nature and severity of the disease state being treated and the mammal undergoing treatment, and is ultimately at the discretion of the physician. A suitable dose of an active ingredient is 1.5 mg to 500 mg for topical administration, the most preferred dosage being 1 mg to 100 mg, for example 5 to 25 mg administered two or three times daily.
By topical administration is meant non-systemic administration and includes the application of the active ingredient externally to the epidermis, to the buccal cavity and instillation of such a compound into the ear, eye and nose, and where the compound does not significantly enter the blood stream. By systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. The active ingredient may comprise, for topical administration, from 0.001% to 10% wlw, e.g. from 1% to 2% by weight of the formulation although it may comprise as much as 10% w/w but preferably not in excess of 5% w/w and more preferably from 0.1% to 1% w/w of the formulation.
The topical formulations of the present invention, both for veterinary and for human medical use, comprise an active ingredient together with one or more acceptable carrier(s) therefor and optionally any other therapeutic ingredient(s). The carrier(s) must be xe2x80x98acceptablexe2x80x99 in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous or alcoholic solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100xc2x0 C. for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
The active ingredient may also be administered by inhalation. By xe2x80x9cinhalationxe2x80x9d is meant intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques. The daily dosage amount of the active ingredient administered by inhalation is from about 0.1 mg to about 100 mg per day, preferably about 1 mg to about 10 mg per day.
In one aspect, this invention relates to a method of treating asthma and a topic disorders (for example, a topic dermatitis and allergies), COPD, rheumatoid arthritis, sarcoidosis and other fibrotic diseases, atherosclerosis, psoriasis, autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, and HIV infection, all in mammals, preferably humans, which comprises administering to such mammal an effective amount of a CCR5 receptor modulator, in particular, a compound as depicted in formula (I).
By the term xe2x80x9ctreatingxe2x80x9d is meant either prophylactic or therapeutic therapy. Such formula (I) compound can be administered to such mammal in a conventional dosage form prepared by combining the formula (I) compound with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The formula (I) compound is administered to a mammal in need of treatment for CCR5-mediated diseases in an amount sufficient to decrease symptoms associated with these diseases. The route of administration may be oral or parenteral.
The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, intra-rectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. The daily parenteral dosage regimen will preferably be from about 30 mg to about 300 mg per day of active ingredient. The daily oral dosage regimen will preferably be from about 100 mg to about 2000 mg per day of active ingredient.
It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a formula (I) compound will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of the formula (I) compound given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
The compounds of formula (I) can be prepared by art-recognized procedures from known or commercially available starting materials. If the starting materials are unavailable from a commercial source, their synthesis is described herein, or they can be prepared by procedures known in the art.
Specifically, compounds of formula (I) wherein R3 is hydrogen are prepared by methods known to the art. For example, in Scheme 1, resorcinol (1-1) and 3,3-dimethylacrylic acid are treated with a suitable reagent, such as sulfuric acid, polyphosphoric acid or boron trifluoride etherate, at a suitable temperature, such as 25-100xc2x0 C., for a suitable time, such as overnight, to give chromanone 1-2. The phenolic hydroxyl group in 1-2 is protected by reaction with a suitable alkylating agent, such as benzyl bromide, in the presence of a suitable base, such as potassium carbonate, in a suitable solvent, such as acetone, at a suitable temperature, such as reflux, for a suitable time, such as 22 hours, to give 1-3. 1-3 is reacted with a suitable organometallic reagent, such as with phenyl Grignard or a substituted phenyl Grignard reagent in a suitable solvent, such as ether, at a suitable temperature, such as reflux, for a suitable time, such as 16 hours, to give the substituted chromene 1-4. The double bond in chromene 1-4 is reduced and the protecting benzyl group removed to give substituted chroman 1-5 by treatment with hydrogen and a suitable catalyst, such as palladium-on-carbon, in a suitable solvent, such as ethanol, at a suitable temperature, such as room temperature, for a suitable time, such as 3 hours. Chroman 1-5 is converted to the benzo-dipyranone 1-6 by treatment with 3,3-dimethylacrylic acid and a suitable agent, such as boron trifluoride etherate, sulfuric acid or polyphosphoric acid, at a suitable temperature, such as room temperature, for a suitable time, such as overnight. Benzo-dipyranone 1-6 is converted to benzo-dipyranamine 1-7 by treatment with a suitable amine, such as methylamine, in the presence of a suitable catalyst, such as titanium tetrachloride, in a suitable solvent, such as benzene, at a suitable temperature, such as room temperature, for a suitable time, such as three days, followed by treatment with a suitable reducing agent, such as sodium borohydride, in a suitable solvent, such as methanol, at a suitable temperature, such as room temperature, for a suitable time, such as one hour. 
a) 3,3-dimethylacrylic acid; H2SO4; b) benzyl bromide, K2CO3, acetone; c) bromo-X-benzene, Mg, ether: d) H2, 10% Pd/C, EtOH; e) 3,3-dimethylacrylic acid, BF3 etherate; f) HNR4R5, TiCl4, benzene; NaBH4, MeOH
Compounds of formula (I) where R3 is hydroxyl are prepared by methods known to the art. For example, in Scheme 2, compound 1-6 is converted to the benzodipyranol 2-1 by reduction of the ketone with a suitable reagent, such as lithium aluminum hydride, in a suitable solvent, such as ether, at a suitable temperature, such as at reflux. Compound 2-1 is dehydrated with a suitable reagent, such as hydrochloric acid, to give the dihydro-benzodipyran 2-2. Compound 2-2 is converted to benzodipyran oxide 2-3 by treatment with a suitable reagent, such as iodine, in a suitable solvent, such as dioxane, followed by treatment with a suitable reagent, such as silver oxide. The epoxide 2-3 is converted to the hydroxy-benzodipyranarnine 24 by treatment with a suitable reagent, such as dimethylamine, in a suitable solvent, such as ethanol, at a suitable temperature, such as room temperature, for a suitable time, such as two days. 
a) LiAIH4, xcex94, Et2O; H2O; b) aq HCl; c) I2, dioxane; Ag2O; d) HNR4R5, EtOH
Compounds of formula (I) where  is a double bond are prepared by the method of Scheme 1, except substituting 1-5xe2x80x2, prepared as described in United States Patent 4,080,335, for 1-5. 
CCR5 Receptor Binding Assay
CHO cell membranes (0.25xc3x97106 cell equivalents) derived from CHO cells stably transfected with CCR5 were incubated with 0.3 125I-RANTES in a 96 well plate for 45 min at room temperature (final reaction volume 200 ul). The reaction was terminated by filtration and the filters (GF/C) were washed twelve times with a solution of phosphate buffered saline containing 0.1 % bovine serum albumin and 0.05% NaN3. The radioactivity bound to filters was measured by liquid scintillation spectrometry. Non-specific binding was determined in the presence of unlabelled RANTES (10 or 30 nM) and averages 30-50% of total binding.
CCR5 Receptor Functional Assay
The cellular functional assay used to assess antagonist activity of compounds was RANTES-induced Ca2+ mobilization in RBL 2H3 cells stably expressing the hCCR5 receptor (RBL 2H3 hCCR5). Agonist activity is determined by Ca2+ mobilization in the same cells which is inhibitable by a selective CCR5 antagonist. In particular, one or more of isomers 1, 2, 3, and 4 demonstrated agonist activity. Cells were grown to 80-100% confluency in T-150 flasks and washed with phosphate-buffered saline. Cells were lifted from the flasks by treating with 3 mL of 1 mM EDTA for 3 min at room temperature and diluting to 2xc3x97106 cells/mL with Krebs Ringer Henseleit buffer (KRH; 118 mM NaCl, 4.6 mM KCl, 25 mM NaHCO3, 1 mM KH2PO4 and 11 mM glucose) containing 5 mM HEPES (pH 7.4), 1 mM CaCl2, 1 mM MgCl2 and 0.1% BSA and centrifuged at 200g for 3 min. Cells were resuspended at 2xc3x97106 cells/mL in the same buffer with 2 xcexcM Fura-2AM, and incubated for 35 min at 37xc2x0 C. Cells were centrifuged at 200xc3x97g for 3 min and resuspended in the same buffer without Fura-2AM, then incubated for 15 min at 37xc2x0 C. to complete the hydrolysis of intracellular Fura-2AM, and then centrifuged as before. Cells (106 cells/mL) were resuspended in cold KRH with 5 mM HEPES (pH 7.4), 1 mM CaCl2, 1 MM MgCl2 and 0.1% gelatin and maintained on ice until assayed. For antagonist studies, aliquots (2 mL) of cells were prewarmed at 37xc2x0 C. for 5 min in 3 mL plastic cuvettes and fluorescence measured in a fluorometer (Johnson Foundation Biomedical Group, Philadelphia, Pa., USA) with magnetic stirring and temperature maintained at 37xc2x0 C. Excitation was set at 340 nm and emission set at 510 nm. Various concentrations of antagonists or vehicle were added and fluorescence monitored for xcx9c15 sec to ensure that there was no change in baseline fluorescence, followed by the addition of 33 nM RANTES. Maximal Ca2+ attained after 33 nM RANTES stimulation was calculated as described by Grynkiewicz et al., (1985). The percent of maximal RANTES-induced Ca2+ was determined for each concentration of antagonist and the IC50, defined as the concentration of test compound that inhibits 50% of the maximal 33 nM RANTES response, obtained from the concentration-response curves (5-7 concentrations of antagonists).
The compounds of this invention show CCR5 receptor modulator activity having IC50 values in the range of 0.0001 to 100 xcexcM. The full structure/activity relationship has not yet been established for the compounds of this invention. However, given the disclosure herein, one of ordinary skill in the art can utilize the present assays in order to determine which compounds of formula (I) are modulators of the CCR5 receptor and which bind thereto with an IC50 value in the range of 0.0001 to 100 xcexcM.